Microbiology (2006), 152, 2515–2528
Review
DOI 10.1099/mic.0.28982-0
Rho-dependent terminators and transcription
termination
M. Sofia Ciampi
Correspondence
M. Sofia Ciampi
Dipartimento di Genetica e Microbiologia, Università di Bari, Via Amendola 165/A, 70126 Bari,
Italy
[email protected]
Rho-dependent transcription terminators participate in sophisticated genetic regulatory
mechanisms, in both bacteria and phages; they occur in regulatory regions preceding the coding
sequences of genes and within coding sequences, as well as at the end of transcriptional units,
to prevent readthrough transcription. Most Rho-dependent terminators have been found in enteric
bacteria, but they also occur in Gram-positive bacteria and may be widespread among bacteria.
Rho-dependent termination requires both cis-acting elements, on the mRNA, and trans-acting
factors. The only cis-acting element common to Rho-dependent terminators is richness in rC
residues. Additional sequence elements have been observed at different Rho termination sites.
These ‘auxiliary elements’ may assist in the termination process; they differ among terminators,
their occurrence possibly depending on the function and sequence context of the terminator.
Specific nucleotides required for termination have also been identified at Rho sites. Rho is the main
factor required for termination; it is a ring-shaped hexameric protein with ATPase and helicase
activities. NusG, NusA and NusB are additional factors participating in the termination process.
Rho-dependent termination occurs by binding of Rho to ribosome-free mRNA, C-rich sites being
good candidates for binding. Rho’s ATPase is activated by Rho–mRNA binding, and provides the
energy for Rho translocation along the mRNA; translocation requires sliding of the message into
the central hole of the hexamer. When a polymerase pause site is encountered, the actual
termination occurs, and the transcript is released by Rho’s helicase activity. Many aspects of this
process are still being studied. The isolation of mutants suppressing termination, site-directed
mutagenesis of cis-acting elements in Rho-dependent termination, and biochemistry, are and will be
contributing to unravelling the still undefined aspects of the Rho termination machinery. Analysis
of the more sophisticated regulatory mechanisms relying on Rho-dependent termination may be
crucial in identifying new essential elements for termination.
Introduction
Bacteria and their phages use two main modes of terminating transcription: Rho-independent or ‘intrinsic’ termination, mainly requiring elements located on the mRNA,
and Rho-dependent termination, relying on both mRNA
elements and trans-acting factors (reviewed by Richardson
& Greenblatt, 1996). Rho-dependent termination has been
intensively studied, and it has previously been extensively
reviewed (Richardson, 2002, 2003). About half of the
transcription terminators identified in Escherichia coli are
Rho-dependent. These terminators lie at the natural end of
genes or within cistrons and in control regions preceding the
coding sequences of genes, where they play an important
role in the regulation of gene expression.
Rho is a homohexameric protein with RNA-dependent
ATPase and helicase activities (reviewed by Platt &
Richardson, 1992), which binds to the mRNA. Essential
sites on the mRNA are the Rho-binding site, known as the
0002-8982 G 2006 SGM
Rho utilization site (rut), and a distal region where the
transcripts are terminated at multiple transcription stop
points (tsp) and released, mediated by Rho interaction with
RNA polymerase (reviewed by Richardson & Greenblatt,
1996). This process requires a ribosome-free transcript of
at least 85–97 nt (Hart & Roberts, 1994). This transcript
exhibits a high-C low-G content, which is consistent with
the known requirements for rC residues and for an unstructured RNA by the Rho activities (reviewed by Richardson &
Greenblatt, 1996; for a summary see Guérin et al., 1998).
Rho loads onto the mRNA at a binding site of 70–80 nt
(Zhu & von Hippel, 1998a), smaller than the ribosome-free
transcript required for the termination process. The Rhobinding site seems to be the primary and essential determinant for Rho termination activity (Guérin et al., 1998).
Thus, the C richness of Rho site sequences could very well be
the main requirement for Rho binding, consistent with the
fact that this is the only feature common to Rho-dependent
terminators.
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M. S. Ciampi
In addition to C richness, other sequence elements, termed
‘auxiliary elements’ in this review, have been observed only
at some Rho-dependent termination sites. For instance,
rutA and rutB elements have been described in the ltR1
terminator (Chen & Richardson, 1987), and suggested to
provide a strong attachment site for Rho (Graham &
Richardson, 1998) on the mRNA. Similar rut elements have
been found at the trp t9 terminator of E. coli (Zalatan et al.,
1993). Some Rho-dependent terminator regions show
sequences with homology to boxA (Friedman & Olson,
1983), which has an antitermination function in the bacteriophage l (Das, 1992) and in other systems (Heinrich
et al., 1995; Vogel & Jensen, 1995); interestingly, boxA-like
sequences in the tna operon of E. coli (Gong & Yanofsky,
2002) and in the his operon of Salmonella typhimurium
(Ciampi et al., 1989; Carlomagno & Nappo, 2001) seem to
be required for Rho-dependent termination. These and
other potential ‘auxiliary elements’ (listed in Table 1),
which are not common features of Rho-dependent terminators, could be part of a pool of elements, different combinations of which may contribute to Rho loading efficiency
and to the efficiency of termination. Which ‘auxiliary
element(s)’ occur at a specific Rho site may depend on the
‘local’ context of the terminator, and this would explain why
not all terminators exhibit the same elements, other than C
richness.
Rho binding to the message activates Rho’s 59 to 39 ATP
dependent RNA–DNA helicase (Delagoutte & von Hippel,
2003), allowing translocation of Rho along the message,
toward polymerase pause sites. At these key sites interactions
with RNA polymerase (Guarente, 1979) lead to termination
and release of transcripts (Yager & von Hippel, 1987). Rho
protein also interacts with other Rho-dependent termination factors, such as NusG (Sullivan & Gottesman, 1992;
for a summary see Burns et al., 1999), NusA (Schmidt &
Chamberlin, 1984; for a summary see Ingham et al., 1999)
and NusB (Carlomagno & Nappo, 2001), which play an
important role in the release of the terminated transcripts.
In this article I review the features of several Rho-dependent
terminators, with the intent of drawing a picture of the
various contexts in which terminators occur and of
the different roles that they play, in relationship to the
mechanism of Rho-dependent termination and to the regulation of gene expression. I also summarize older and more
recent data on the various elements involved in factordependent termination, emphasizing important insights
that affected this field and remaining problems. Models for
Rho-dependent termination are also discussed.
I start with a survey of Rho-dependent terminators: for
each terminator the facts most relevant to the role of the
terminator and to the mechanism of termination are presented; similarities and differences among the terminators
are highlighted. The terminators are classified into two
groups: constitutive and regulated terminators (see Table 2).
The regulated terminators are divided into two subgroups:
conventionally regulated Rho-dependent terminators and
intragenic terminators, most of the latter being those
responsible for transcriptional polarity effects. The main
focus of the discussion will not be on the regulatory
mechanisms, rather on the features of the Rho-dependent
terminators themselves. I then review cis-acting sequence
elements and trans-acting factors required for Rhodependent termination. The ‘auxiliary elements’ identified
at some terminators are presented and discussed for the
individual terminators. Finally, ideas and facts leading to
the current models for Rho-dependent termination are
summarized.
Constitutive Rho-dependent terminators
Some Rho-dependent terminators occur at the end of transcriptional units, where transcription stops independently
of any regulatory mechanism. As expected, these terminators are constitutive. Three such terminators are described
in this section: the trp and tyrT operon terminators of
E. coli, and the gene IV transcript terminator of the E. coli
bacteriophage f1 (Table 2).
trp operon of E. coli. One of the most investigated
Rho-dependent terminators is trp t9, located at the very
end of the trp operon of E. coli, terminating the synthesis
of the polycistronic trpA–E mRNA (Platt, 1981). In vitro
studies of a DNA fragment carrying the terminator region
revealed that the terminator is Rho-dependent and that
Table 1. ‘Auxiliary elements’ associated with Rho-dependent termination sites
Location of terminator (name)
trp operon of E. coli (trp t9)
tyrT operon of E. coli
cro gene of l (tR0)
Colicin E1 gene of E. coli
Rightward operon of l (tR1)
hisG gene of S. typhimurium
tna operon of E. coli
ilv operon of E. coli
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Auxiliary elements
rutA-like and rutB-like
R1 and R2
rutA and rutB
boxA-like
boxA-like
boxA-like
CAAUCAA
CAACAA
CAAACAA
CAAUCAA
CAAUCGA
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Microbiology 152
Rho-dependent terminators and transcription termination
Table 2. Classification and location of Rho-dependent terminators
Location of terminators (name)
Constitutive terminators
trp operon (trp t9)
tyrT operon
Gene IV
Conventionally regulated terminators
Rightward operon (ltR1)
tna operon
rho gene
rho gene
Intragenic regulated terminators
his operon
trp operon
trp operon
ilv operon
lac operon
gal operon
malK-lamB operon
nifLA operon
Left operon (timm)
Organism
E. coli
E. coli
E. coli phage f1
End of operon
End of operon
End of gene
E. coli phage l
E. coli
E. coli
B. subtilis
Between cro and cII genes
Between leader region and structural
genes
Leader and 59 end regions
Leader and 59 end regions
S. typhimurium
E. coli
B. subtilis
E. coli
E. coli
E. coli
E. coli
K. pneumoniae
E. coli phage P4
Within
Within
Within
Within
Within
Within
Within
Within
Within
the actual sites of termination are spread over a region of
about 100 nt (Wu et al., 1981).
Transcription of the DNA fragment from the gal promoter
showed that the activity of the terminator is not promoterspecific, allowing deletion analysis of the first 105 bp
sequence of the region and identification of a Rho ‘loading
site’ and of a pair of elements within it (summarized by
Zalatan et al., 1993). These elements are examples of what I
have defined above as ‘auxiliary elements’; that is, they are
not common to all terminators, but they are found in the
specific context of one or a few terminators. Indeed, the
pair of elements observed at the trp t9 terminator are
similar to the rutA and rutB sequences identified in the l
tR1 terminator (see later) and proposed to be required for
the ATPase activity of Rho (Chen & Richardson, 1987). In
trp t9, the rutB-like element seems to be more important
than the rutA-like element, which appears to be nonfunctional unless the rutB-like sequence is present. Two
similar subelements, R1 and R2, within the Rho-loading site,
have also been proposed to be important for Rho-dependent
termination at trp t9 (Zalatan et al., 1993).
Efficient loading of Rho at trp t9 guarantees efficient and
precise termination at the downstream termination zone; in
fact, introduction of a secondary structure within the Rho
loading site both reduces termination efficiency and shifts
the initial termination site downstream (Zhu & von Hippel,
1998a). Zhu & von Hippel (1998a) also showed that
approximately 97 nt of transcript are required by trp t9 to
cause termination. They also found that termination efficiency depends on the sequence context at the 39 end of the
transcripts; the transcript terminus probably affects the
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Position of terminators
hisG and hisC coding regions
trpE coding region
trpE coding region
ilvGM region
lacZ coding region
galE coding region
lamB coding region
nifA coding region
kil coding region
interactions of components of the transcription complex
with the transcription bubble and thereby affects pausing.
Indeed, the efficiency of RNA release at the individual
termination positions is controlled by a kinetic competition
between RNA polymerase elongating the transcripts and
Rho terminating them (Zhu & von Hippel, 1998b). These
authors found no requirement for specific sequences, like
the previously identified rut sites, under the conditions used
in their experiments, which is consistent with the view
that Rho loading occurs at sites that are simply devoid of
secondary structures and that contain a number of rC
residues compatible with the activation of the RNAdependent ATPase of Rho.
tyrT operon of E. coli. Transcription termination of the
tyrT operon of E. coli, encoding tRNATyr, occurs at a
Rho-dependent terminator, both in vivo (Rossi et al.,
1981) and in vitro (Küpper et al., 1978), and is completely
dependent on Rho-factor. The site of termination is A?T
rich and the adjacent sequences on both sides are G?C
rich (Küpper et al., 1978). An A?T rich region preceded
by a small cluster of G?C pairs is common to some other
Rho-dependent terminators, thought to cause a pause in
transcription downstream. Small hairpin structures have
been found at some termination sites, just before the
end of the transcript, but they are absent at the tRNATyr
terminator (Küpper et al., 1978). On the other hand, the
potential ‘auxiliary sequence’ CAAUCAA, observed at
some Rho sites (see later), is also found at the tyrT operon
terminator (Küpper et al., 1978). Madden & Landy (1989)
showed that progressive removal of the region preceding the
tRNATyr terminator progressively reduces Rho-dependent
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M. S. Ciampi
termination activity, consistent with the idea that upstream sequences mediate the efficiency of Rho-dependent
termination.
Gene IV of bacteriophage f1. A peculiar type of termi-
nator lies at the end of gene IV of the E. coli bacteriophage
f1. Readthrough transcription at this terminator is
observed in nonsense suppressor or rho hosts (Moses &
Model, 1984; La Farina et al., 1990), indicating that translating ribosomes are responsible for suppression of termination, through occupation of a region of RNA which is
not normally translated. The observations further indicate
that Rho factor is required for the activity of the terminator. The terminator is peculiar in the sense that it seems
to be Rho-dependent, according to the above observations, but not according to other features of the terminator site. Indeed, the region of RNA between the nonsense
codon for translation termination of gene IV and the 39
end of the Rho-terminated transcripts is 63 nt long and
not sufficient to guarantee Rho binding, according to the
minimum size of ribosome-free transcript required for
Rho termination; this suggests that the common concept
of a Rho-binding site needs to be refined. An additional,
unusual feature of this terminator is that the termination
site occurs within a predicted, fairly stable stem–loop
structure (La Farina et al., 1990), in contrast with the
generally accepted idea of the requirement of a mostly
unstructured RNA by the Rho activities. Thus, the context
in which Rho-dependent termination occurs at this site
seems to be quite different from that of other characterized Rho sites. On the other hand, an analysis of the distal
portion of the gene IV coding sequence revealed similarity
with sequences observed in other Rho-terminated transcripts (La Farina et al., 1990).
The ‘auxiliary elements’ or potential ‘auxiliary elements’
found at the constitutive Rho sites described above are not
common to the terminators of this class (Table 1); thus,
these elements don’t seem to be specific to the role of
constitutive terminators as natural terminators or to the
location of these terminators at the end of transcriptional
units. The next section describes Rho-dependent terminators whose activity is regulated.
Regulated Rho-dependent terminators
Most of the Rho-dependent terminators identified and
studied are regulated by various and sophisticated mechanisms. Some show a conventional regulation, requiring
trans-acting factors, and others are regulated by cis-acting
elements, located within the coding regions, the latter being
terminators responsible for natural polarity.
Conventionally regulated Rho-dependent terminators
Rightward operon of bacteriophage l. Rho plays a central role in the life cycle of the E. coli bacteriophage l, as a
key function in the regulation of gene expression. The tR1
terminator of l is one of the best-studied Rho-dependent
terminators. tR1 lies within the major rightward operon
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of l, in the intercistronic region between the cro and cII
genes, and it controls N-mediated expression of the
downstream genes in the operon. Two regions were
identified in l tR1, rut and tsp. rut, in the mRNA, binds
Rho. It was dissected into two elements, rutA and rutB,
separated by the N-utilization element boxB (Chen &
Richardson, 1987). The roles of these ‘auxiliary elements’
were tested in vivo, assaying the effects of deletions of rut
sequences both on the ability of the mutant transcripts to
bind Rho and to activate Rho ATPase and on the overall
activity of the terminator (Graham & Richardson, 1998).
These studies suggested that the rut site guarantees correct
positioning of the RNA with respect to the Rho protein,
for activation of ATPase, in addition to providing a strong
attachment site for Rho. Removal of rutA has a more
dramatic effect on the function of tR1 than does removal
of rutB (Graham & Richardson, 1998). Indeed, rutA RNA
is more C-rich than rutB RNA, consistent with the proposed role of C residues in the interaction of Rho with
the mRNA (McSwiggen et al., 1988) and in the function
of Rho-dependent terminators (Hart & Roberts, 1991;
Zalatan & Platt, 1992). Deletion of the boxB sequence of
the l cro gene, which forms a small stem–loop structure
in the RNA, has no effect on the affinity of the cro RNA
for Rho, nor on the efficiency of termination at tR1
(Graham & Richardson, 1998); this contrasts with the
observation that small hairpin structures are generally
found in transcripts to which Rho binds (Schneider et al.,
1993). Since no differences were observed between the
findings in vivo and those in a purified in vitro system
with Rho factor only (Graham & Richardson, 1998), possible interaction of other factors does not, apparently,
interfere with Rho action in the rut region. The in vitro
results seem to fully account for the rut functions in vivo
(Graham & Richardson, 1998), and suggest that no other
cis-acting sequences outside rut are necessary for tR1
function.
The rutA element carries a sequence with only one mismatch
with the consensus sequence of boxA, a site of interaction
of the host-encoded NusB, NusG and S10 proteins, for
antitermination in bacteriophage l (Mogridge et al., 1998).
Similar ‘auxiliary elements’ have been identified in the his
operon of Salmonella (Ciampi et al., 1989; Carlomagno &
Nappo, 2001) and in the tna operon of E. coli (Gong &
Yanofsky, 2002) (Table 1), where they seem to be required
for Rho-dependent termination.
The other region identified in l tR1, tsp, carries three
heterogeneous RNA stop points, which correspond to
polymerase pause sites (Lau et al., 1983; Richardson &
Richardson, 1996). This region can be substituted with an
alien sequence, carrying polymerase pause sites, without
affecting termination at the transcription stop points
(Richardson & Richardson, 1996), which shows independence of the rut and tsp action in the termination. Termination at one of the RNA stop points of l tR1 occurs at
the sequence CAAUCAA preceded by a potential weak
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Rho-dependent terminators and transcription termination
stem–loop structure in the RNA transcript (Rosenberg et al.,
1978). Similar putative ‘auxiliary elements’ have also been
found at the l tR0 Rho-dependent terminator (CAACAA)
(Calva & Burgess, 1980), near Rho-dependent transcription
stop points within the ilv operon (CAAUCGA) (Wek et al.,
1987) and at the Rho-dependent terminators following the
tRNATyr (CAAUCAA) (Küpper et al., 1978) and the colicin
E1 (CAAACAA) (Ebina & Nakazawa, 1983) genes (Table 1);
other terminators do not have CAAUCAA-like sequences.
Lau et al. (1984) tested the possibility that these features are
sufficient to signal Rho-dependent termination at l tR1, and
found that they are insufficient, suggesting that there are
additional sequence requirements for Rho action.
Specific residues required for Rho–RNA interaction at l tR1
have recently been identified by Graham (2004): single
nucleotide substitutions at eight positions in a 25 nt region,
previously shown to be critical for termination, reduce
termination. These substitutions occur at the 59 end of rutA
and introduce, in most cases, guanosines, confirming Rho’s
idiosyncrasy for Gs; not surprisingly, many substitutions
result in the loss of cytidine residues, confirming Rho’s
‘sympathy’ for Cs.
The l tR1 terminator region shows several ‘auxiliary
elements’ or potential ‘auxiliary elements’ (Table 1), as
mentioned above. This suggests that the terminator may
require the contribution of more elements for its activities
than those needed at other terminator sites. These
requirements may be dictated by the specific location of
the terminator within the rightward operon of l, in relation
to its role of regulator of gene expression.
Some conventionally regulated Rho-dependent terminators
occur in regulatory regions located at the beginning of
transcriptional units. Such terminators have been found in
the tna operon of E. coli and in the rho genes of E. coli and
Bacillus subtilis (Table 2), and they are described below.
tna operon of E. coli. A Rho-dependent terminator,
playing a key role in regulation of expression of the degradative tryptophanase (tna) operon of E. coli (Stewart et al.,
1986), lies between the sequences of the tnaC leader and
of the first structural gene of the operon, tnaA. It prevents
expression of the operon when no tryptophan is available
and termination is inhibited when the inducer tryptophan
is provided. Studies of tna constitutive mutants disclosed
elements that participate in Rho-dependent termination,
mainly a Rho-utilization site (Gollnick & Yanofsky, 1990)
and a sequence with similarity to boxA (Konan &
Yanofsky, 2000). These studies suggest that, in the absence
of inducer, Rho binds to the leader portion of the tna
transcript, moves 59 to 39 along the transcript until it
reaches a paused polymerase, and then drives the elongation complex to terminate transcription. Operon induction requires the presence of a single, crucial, trp codon in
the leader-peptide coding region and requires translation
of tnaC. In the presence of tryptophan, the TnaC-peptidyltRNA remains bound to the translating ribosome, which
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is not released at the tnaC stop codon, preventing Rho
factor access to the boxA and rut sites, and consequently
inhibiting termination (Gong & Yanofsky, 2003). This
ribosome release inhibition appears to be the crucial event
that regulates Rho-dependent termination in the tna
operon leader region.
rho gene of E. coli and B. subtilis. The expression of the
E. coli rho gene itself is regulated through Rho-dependent
termination (Matsumoto et al., 1986). In fact, Rho-dependent
terminators have been identified in the mRNA leader and
N-terminal regions of the rho gene, and they have been
proposed to be responsible for reducing expression of rho
in response to high levels of the coded protein.
Similarly, the rho gene of B. subtilis seems to use Rhodependent terminators for autoregulation of the gene
(Ingham et al., 1999). The ability to regulate Rho levels
may allow the cell to modulate gene expression by using
Rho-dependent terminators strategically located between
genes and their promoters. Interestingly, Rho-dependent
termination also plays a role in trp operon expression of B.
subtilis (see later), these two cases being, to the best of my
knowledge, the only known examples of gene regulation by
Rho-dependent termination in Gram-positive bacteria.
Intragenic regulated Rho-dependent terminators
It has been known for a long time that Rho-dependent
terminators are responsible for premature blocks of
transcription within operons, in response to premature
translation termination or inefficient translation initiation
caused by stress conditions. Such a response is the result
of the fact that transcription and translation are coupled.
These intragenic terminators are cryptic; in fact, they don’t
normally function, but are unmasked only by pathological
conditions interfering with proper translation of polycistronic mRNAs and uncoupling transcription from translation as a consequence. Rho-dependent termination within
operons is responsible for transcriptional polarity effects on
expression of the genes downstream from the transcriptional block; such a mechanism may have evolved to conserve
energy, by preventing the synthesis of unused transcripts
(reviewed by Richardson, 1991). Rho-dependent terminators causing polarity effects have been identified and studied
in a number of systems (Table 2). Several of these terminators are presented below.
his operon of S. typhimurium. In the first gene of the
histidine (his) operon of S. typhimurium, hisG, a Rhodependent terminator, is responsible for the polar effects
caused by point mutations and by insertions of transposable elements Tn10 and Tn5 (Ciampi et al., 1982;
Ciampi & Roth, 1988; Wang & Roth, 1988). This terminator was identified by the isolation and characterization of
mutants suppressing the strongly polar hisG frameshift
mutation hisG2148 (Ciampi et al., 1982; Ciampi & Roth,
1988).
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In vivo analysis of the transcripts produced by the polar
mutant unveiled the existence of five major transcription
end points in hisG, all affected by Rho to a different extent
(Ciampi et al., 1989). Three of these sites fall immediately
downstream of sequences with the potential for forming
secondary structures (Ciampi et al., 1989) and one of them,
the first in the gene and the most affected in a rho background, occurs within a sequence (Ciampi et al., 1989)
resembling the boxA consensus element. This Salmonella
his sequence, cloned on a plasmid, was shown to enhance
termination by interacting with the NusB factor of E. coli
cells (Carlomagno & Nappo, 2001). Salmonella hisG
sequences required for premature, Rho-dependent termination of transcription (Ciampi & Roth, 1988; Ciampi et al.,
1989) were also cloned on a plasmid and used to show that
NusA modulates this intragenic termination, in nusA E. coli
hosts (Carlomagno & Nappo, 2003). Three of the termination sites in hisG are also weakly active in the normally
translating wild-type strain, similarly to what was observed
in the Salmonella hisC cistron (Alifano et al., 1988) and in
the E. coli lacZ gene (Stanssens et al., 1986). These sites may
coincide with polymerase pause sites, as described in other
systems (Yager & von Hippel, 1987).
The histidine operon of S. typhimurium proved particularly
useful for the isolation and study of polarity suppressor
mutants. Several mutations affecting the hisG terminator
have been isolated, all of which are deletions, suggesting that
no sequence-specific element is required for Rho-dependent
termination. Indeed, four point mutations scattered along
a stretch of 18 bp removed by the smallest of the deletions
do not damage the terminator (Ciampi & Roth, 1988). Two
of the deletions do not remove any of the five transcription
end-points within hisG (G. Lisanti & M. S. Ciampi,
unpublished results) and they may identify a binding site
for Rho factor (Ciampi & Roth, 1988). Other deletions
show that progressive removal of the sequence including
the transcription stop points has an increasing suppressing effect on polarity, leading to progressive increase of
downstream gene expression (G. Lisanti & M. S. Ciampi,
unpublished results). Mutations suppressing polarity by
reinitiation of transcription (Bianco et al., 1998) or of
protein synthesis (M. R. Pasca, A. La Teana & M. S. Ciampi,
unpublished results) within hisG were also isolated, consistent with transcription terminating in hisG in polar
mutants and with the known interference of translation on
Rho-dependent termination.
A cryptic Rho-dependent terminator is also responsible for
premature termination of transcription in the hisC cistron
of S. typhimurium (Alifano et al., 1988), where polarity
suppressor mutations affecting initiation of translation or
transcription have been isolated and studied (M. R. Pasca &
M. S. Ciampi, unpublished results). Several 39 transcription
end points were mapped within the hisD gene, immediately
following hisG in the operon, and polar hisA mutants also
produce shorter mRNAs (Alifano et al., 1991), but possible
action of Rho at these sites remains to be defined. Polar
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effects are also caused by mutations in the three remaining
internal genes of the his operon, hisB, hisH and hisF (Ciampi
et al., 1982); hence, cryptic Rho sites may be embedded in
the coding sequences of these cistrons as well. In vivo studies
with polarity suppressor mutants, including rho mutants,
and in vitro transcription experiments with purified Rho on
the hisD, hisB, hisH, hisA and hisF cistrons, would provide a
complete picture of the distribution and features of Rhodependent termination sites in the his operon. The scenery
of the his operon could be common to most, if not all,
bacterial operons; indeed, most operons may have evolved
such a mechanism to cope with translation pathology.
trp operon of E. coli and B. subtilis. The first gene of
the trp operon of E. coli, trpE, also shows intragenic Rhodependent termination, which is the cause of polarity
effects (Korn & Yanofsky, 1976). Investigation of trpE
revealed several termination sites and some pause sites
within the coding sequence of the gene; however, the
study of Rho-dependent termination in the trp operon
did not focus on the features of the terminator, but rather
on the ability of the polynucleotide-binding sites of the
Rho protein to interact with the mRNA. This was investigated in rho mutants showing increased polarity in the trp
and lac operons, by Tsurushita et al. (1989), who found
that the primary Rho-binding site directs the efficiency of
termination, while the secondary site selects the termination points.
Rho-dependent termination plays a sophisticated role in the
expression of the trpE–A operon of B. subtilis, the mechanism involving transcription attenuation and translational
control. Translational control relies on the formation of
an RNA hairpin that sequesters the trpE Shine–Dalgarno
(SD) sequence, blocking translation. Formation of the SDblocking hairpin causes transcriptional polarity, by allowing
Rho’s access to the nascent transcript, downstream from this
RNA structure (Yakhnin et al., 2001).
ilv operon of E. coli. Rho-dependent termination is
responsible for transcriptional polarity in the ilv operon
of E. coli K-12. Three Rho-dependent termination sites
were identified and partially characterized by in vitro transcription experiments with a restriction fragment containing the promoter-distal portion of ilvG and the proximal
portion of ilvM, fused to the tac promoter (Wek et al.,
1987). Several sequences with no dyad symmetry, required
for Rho interaction with the mRNA, were identified: two
of these sequences fall immediately upstream of two of
the termination sites (Wek et al., 1987). Regions with
regularly spaced cytidine residues, observed in other Rhodependent terminators (Platt, 1986), lie upstream of the
transcription stop points in the ilv9GM9 region, and
the sequence CAAUCAA, found adjacent to some Rhodependent termination sites (Lau et al., 1984) (Table 1),
is also present, with one mismatch (CAAUCGA), near the
three transcription stop points in the ilv9GM9 region (Wek
et al., 1987); however, a possible role for these potential
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Rho-dependent terminators and transcription termination
‘auxiliary elements’ in Rho-dependent termination remains
to be established.
lac and gal operons of E. coli. Inefficient translation
initiation causes premature transcription termination in
the first gene of the lac operon of E. coli, lacZ (Stanssens
et al., 1986). Four cryptic Rho-dependent termination
sites, which are turned on by upstream blocks of protein
synthesis, as well as by amino acid starvation, were identified in lacZ, by in vitro and in vivo studies; they correspond to polymerase pause sites (Ruteshouser & Richardson,
1989). A detailed analysis of the sequence showed a complex interplay of polymerase-pause and terminator elements, widely discussed by these authors. Two of these
terminators in the early region of lacZ (tiZ1 and tiZ2)
proved particularly useful in testing the requirement for
the NusG and NusA factors in Rho-dependent termination: NusG has a stimulating effect on Rho-dependent termination in laZ, while NusA inhibits termination (Burns
et al., 1998) (see section on trans-acting factors).
Cryptic Rho-dependent terminators responsible for polarity
effects also lie in the gal operon of E. coli (De Crombrugghe
et al., 1973). The termination sites have not been identified
and characterized at the molecular level, but this system has
mostly been used to study the requirement for the E. coli
NusG protein in Rho-dependent termination. Rho-induced
polarity in the gal operon is suppressed in NusG-deficient
cells and Rho-dependent termination in galE, the first gene
of the operon, is entirely prevented upon NusG depletion
(Sullivan & Gottesman, 1992) (see also section on transacting factors).
malK-lamB operon of E. coli. The presence of a Rho-
dependent terminator has been inferred within the lamB
gene of the malK-lamB operon of E. coli K-12 (Colonna &
Hofnung, 1981). Expression of lamB, the structural gene
for the l receptor (Thirion & Hofnung, 1972), results in
phage sensitivity. Expression of the operon requires an
activator, the malT gene product. A malT mutant is resistant to l, since lamB is not expressed; however, in the
absence of the malT gene product, expression of lamB
and sensitivity to l infection are restored in a rho mutant,
due to the activity of a cryptic promoter, located at the
distal end of malK and unmasked in the mutant rho
(Colonna & Hofnung, 1981). Transcription originating at
this promoter is terminated by Rho at the inferred terminator within lamB, in a malT rho+ strain, and this prevents expression of lamB. The terminator’s structure and
mechanism of function remain to be explored.
Intragenic Rhodependent termination has been studied in the nifLA
operon of K. pneumoniae, encoding the sensor–activator
pair involved in the regulation of other nif genes. In this
operon, altered translation of nifL activates Rho-dependent
termination not in that gene, but in the downstream nifA
(Govantes & Santero, 1996), which has been explained by
nifLA operon of Klebsiella pneumoniae.
http://mic.sgmjournals.org
the fact that L and A are translationally coupled (Govantes
et al., 1998). These studies provide new insight into the
putative role of regions of biased C and G composition in
Rho-dependent termination. An analysis of transcripts
terminated within the his operon, as well as in the lacZ
cistron and at the Rho-dependent trp t9 and l tR1 terminators, led Alifano et al. (1991) to hypothesize that a
region of high cytosine-over-guanosine content in the
mRNA is an essential feature of Rho-dependent terminators. Such C>G regions were also found at the nifLA
operon’s terminators; however, other C>G regions in the
operon do not show termination activity (Govantes &
Santero, 1996). Hence, a direct correlation between the
occurrence of C>G regions and Rho-dependent termination could not be established by these authors, who suggest that the distribution of cytosines, yielding specific
C/G profiles, may only be important in diagnosing the
possible presence of a terminator.
Rho-dependent termination within coding sequences is not
in all cases triggered by pathological conditions interfering
with proper mRNA translation; in fact, intragenic termination can also be part of a physiological regulation of gene
expression. One such case has been reported in the left
operon of the E. coli bacteriophage P4.
Left operon of P4. A Rho-dependent terminator is
involved in the establishment of P4 lysogeny, through a
positive feedback mechanism on termination. The terminator, named timm, occurs within the coding sequence of
the first translated gene, kil, of the left operon for replication of P4 and it seems to participate, with two upstream
Rho-independent terminators, to attenuate expression of
the left operon functions, early after infection (Briani
et al., 2000). Translation of kil inhibits termination at
timm (Forti et al., 1999), which may contribute to the
production of a leader transcript, which is matured into a
small, untranslated RNA, CI, a phage factor required for
efficient termination. Once produced, CI acts as an antisense RNA, on the RNA leader of the left operon, where
RNA–RNA interactions are essential for stringent termination at the downstream timm (Briani et al., 2000),
which in turn prevents expression of the replication
genes and guarantees immunity against superinfecting
phages.
Summary of Rho-dependent terminators
Some of the regulated Rho-dependent terminators described
above lie in regulatory regions separating genes from their
promoters, which may provide the cell with a convenient
mechanism for the control of gene expression. Most regulated Rho-dependent terminators occur within coding
sequences, contributing to preventing wasteful expression
of genes, but this location is also used to direct gene
expression. Rho-dependent termination can also serve to
end transcriptional units; however, this role seems to be
minor among the cases identified so far.
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M. S. Ciampi
Looking back over all Rho-dependent termination sites,
their features don’t seem to depend on the common location
or role of the terminators. ‘Auxiliary elements’ and potential
‘auxiliary elements’, observed at some of the Rho sites, do
not appear to be specific to a specific class of terminators
(Tables 1 and 2). These ‘auxiliary elements’ could be part of
a pool of elements, one of which or combinations of which
may contribute to Rho loading efficiency and to the overall
efficiency of termination. The requirement for and effect on
termination could be dictated by the ‘local’ context of the
individual terminators, and this would explain why not all
terminators exhibit the same ‘auxiliary elements’. How the
individual sequence elements contribute to the structure
of Rho-dependent terminators and to the termination
mechanism is not fully understood yet. Analysis of the more
sophisticated mechanisms relying on Rho-dependent termination may be crucial in identifying new essential
elements of the Rho-dependent termination machinery.
The isolation of mutants relieving transcriptional polarity,
site-directed mutagenesis of the various sequence elements,
and biochemistry, will contribute to uncovering the secrets
of Rho-dependent termination.
Cis-acting features of Rho-dependent
terminators
Various cis-acting elements have been observed at different
Rho-dependent termination sites and they are discussed, for
the individual Rho sites, in the section on Rho-dependent
terminators. However, the only feature common to Rhodependent terminators seems to be richness of rC residues
in the mRNA. In fact, the strongest activators of NTP
hydrolysis by Rho are poly(rC) and rC-rich oligonucleotides. The requirements for rC residues by the Rho activity
have been extensively studied (reviewed by Richardson &
Greenblatt, 1996; summarized by Guérin et al., 1998): sitedirected mutagenesis of the rut site of the trp t9 terminator
did not identify a number or distribution of rC residues that
could be directly correlated with the efficiency of termination (Zalatan & Platt, 1992). Recently, however, Graham
(2004) showed that single nucleotide substitutions in the
rutA region of ltR1, which reduce termination, affect C
residues and most of them introduce Gs. The C/G ratio of
Rho-dependent terminators has been proposed to be an
essential feature for termination (Alifano et al., 1991), but
it may only be indicative of the presence of a terminator
(Govantes & Santero, 1996). Short rUrC oligomers stimulate Rho’s ATPase more than do rC homo-oligomers (Wang
& von Hippel, 1993); besides, a 30 nt run of pure rUrC,
introduced between a promoter and a non-terminator site,
is sufficient to cause transcription termination (Guérin et al.,
1998). This termination is dependent on the Salmonella Rho
factor (identified by Housley & Whitfield, 1982), and is
observed only in the absence of protein synthesis, consistent
with the known incompatibility of Rho action with translation and with studies of Rho-dependent termination in the
hisG gene of Salmonella (Ciampi et al., 1982; Ciampi &
Roth, 1988). According to the results of Guérin et al. (1998),
2522
Rho-dependent termination seems to depend essentially on
the Rho loading site.
Trans-acting factors in Rho-dependent
termination
Rho factor. The hexameric protein Rho factor constitutes
0?1 % of the total soluble protein of E. coli and S. typhimurium (Housley & Whitfield, 1982); it is an essential
protein for the growth of E. coli and of its lambdoid
phages (Das et al., 1976), for Rhodobacter sphaeroides
(Gomelsky & Kaplan, 1996) and for Micrococcus luteus
(Nowatzke et al., 1997); however, Rho is dispensable in B.
subtilis (Quirk et al., 1993) and in Staphylococcus aureus
(Washburn et al., 2001). Analysis of bacterial sequences
suggests that rho-like homologues are ubiquitous in the
bacteria and highly conserved (Opperman & Richardson,
1994; Washburn et al., 2001).
Rho acts as a 59 to 39 ATP dependent RNA–DNA helicase,
triggered by the interaction with the mRNA at the Rho
loading site (reviewed by Richardson, 2003, and by
Delagoutte & von Hippel, 2003). ATP hydrolysis provides
the free energy for Rho translocation 59 to 39 along the
message, until the protein encounters the transcription
elongation complex stalled at a pause site. At these sites
transcription is terminated and the RNA is dissociated
from the transcription complex, by Rho’s 59 to 39 helicase,
which disrupts the transcript–template duplex, again
powered by ATP hydrolysis.
Much of Rho’s structure and function were known
(reviewed by Richardson, 2002), prior to the crystallographic studies of Skordalakes & Berger (2003). Rho
functions as a homohexameric ring (Finger & Richardson,
1982; Geiselmann et al., 1992). Each Rho monomer bears
two RNA-binding domains, the so-called primary and
secondary sites (Richardson, 1982), located on the smaller
N-terminal and larger C-terminal domains, respectively.
Skordalakes and Berger’s work shows that the two RNAbinding sites, the primary one, responsible for mRNA
recognition, and the secondary one, required for translocation and unwinding, point toward the centre of the ring,
and that the complexed hexameric ring is split open (likened
to a ‘lock washer’), a configuration likely required to allow
Rho loading onto mRNA and translocation (reviewed by
Richardson, 2003). Jeong et al. (2004), analysing the conformational changes of the Rho protein following nucleotide and nucleic acid binding, concluded that RNA binding
changes the conformation of the Rho hexamer, resulting in
opening of the subunit interfaces. ATP binding occurs and
a new conformational change takes place, following RNA
contact with the secondary sites lining the central channel of
the Rho ring, which makes Rho capable of hydrolysing ATP
and translocating.
NusG factor. Rho factor is sufficient to terminate tran-
scription in vitro at most Rho-dependent terminators,
whereas, in vivo, an additional factor, NusG, is required
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Rho-dependent terminators and transcription termination
by several Rho-dependent terminators. NusG is an essential, abundant protein in E. coli (Li et al., 1992), whereas
in B. subtilis NusG is not essential either for viability or
for the autoregulation of Rho (Ingham et al., 1999). NusG
was originally isolated as a factor aiding the N protein in
antitermination of transcription in bacteriophage l (Li
et al., 1992); it is a transcription elongation factor, which
stimulates the rate of transcription (Burova et al., 1995)
and enhances suppression of class II polymerase pause
sites (Artsimovitch & Landick, 2000). Orthologue nusG
genes exist in all the bacteria whose genomes have been
sequenced, including Mycoplasma genitalium, which lacks
other important transcription factors (Fraser et al., 1995).
The requirement for NusG in Rho-dependent termination
was first studied by Sullivan & Gottesman (1992), who
showed that cells depleted of NusG have reduced efficiency
of transcription termination at certain Rho-dependent
terminators. However, some sites are fully dependent on
NusG: Rho-dependent termination in galE, the first gene of
the gal operon, is entirely prevented upon NusG depletion
(Sullivan & Gottesman, 1992). In vitro, NusG is strongly
required at the tiZ1 terminator and its requirement is
context dependent at tiZ2 (Burns et al., 1999). NusG does
not greatly affect in vitro termination at the trp t9 (Nehrke
et al., 1993) nor at the l tR1 (Burns et al., 1999) terminators;
however, in vivo, the l tR1 terminator is strongly dependent
on NusG (Sullivan & Gottesman, 1992). The discrepancy
between in vitro and in vivo requirements at l tR1 may be
due to the more-limiting context for Rho action in vivo,
where the presence of ribosomes translating the message
creates more pressure on Rho’s interaction with the mRNA.
In fact, at tiZ2 and l tR1 terminators NusG is required to
overcome kinetic limitations that may be imposed on Rho
function, depending on the properties of the specific terminator and on the position of the terminator in the transcriptional unit (Burns et al., 1999); this requirement may
involve NusG-mediated modulation of the sensitivity of the
transcription complex to Rho action. Alternatively, the
NusG effect at l tR1 in vivo could be the consequence of its
involvement in other events important for Rho activity,
such as ribosome release. Indeed, nusG orthologues have
been found in two organisms that lack Rho, Mycoplasma
genitalium (Fraser et al., 1995) and Methanococcus jannaschii
(Bult et al., 1996), which suggests a NusG role other than in
Rho-dependent termination.
NusG has been suggested to connect Rho to the RNA
polymerase–RNA complex, thereby facilitating recognition
of the termination signals (summarized by Burns et al.,
1999; reviewed by Richardson, 2002). Besides, the interaction of NusG with the RNA polymerase may cause this
enzyme to become more susceptible to Rho actions that
mediate release of the transcripts, as proposed by Burns et al.
(1999), who have shown that NusG enhances the rate of
release of the RNA from the transcription elongation
complex. The NusG effect on release may also be the cause of
the requirement for NusG at some terminators.
http://mic.sgmjournals.org
NusA factor. NusA has been reported to stimulate Rhodependent termination (Kainz & Gourse, 1998); however,
in most cases it inhibits Rho (Kainz & Gourse, 1998),
consistent with the involvement of NusA with nut utilization in l antitermination (Li et al., 1992) and with rRNA
antitermination (Vogel & Jensen, 1997). NusA is an essential protein in E. coli (Kainz & Gourse, 1998), but it
becomes dispensable in rho mutants with reduced termination efficiency (Zheng & Friedman, 1994), indicating a
critical role in Rho-dependent termination. In B. subtilis
NusA is essential; however, its requirement is independent
of Rho (Ingham et al., 1999), which suggests the involvement of the protein in some process other than Rhodependent termination. As for NusG, NusA orthologues
exist in all the bacteria whose chromosomes have been
sequenced.
NusA binds both to Rho factor and to E. coli RNA
polymerase, which provides a way of coupling Rho to the
elongation complex (Schmidt & Chamberlin, 1984). The
Rho–NusA interaction may modulate Rho action at certain
terminators. NusA is also a transcription elongation factor,
which, unlike NusG, slows transcript elongation, by enhancing RNA polymerase pausing at some pause sites (Lau et al.,
1983; Artsimovitch & Landick, 2000). Zheng & Friedman
(1994) have proposed that the ability of NusA to enhance
pausing, which results in tight coupling of transcription
and translation, would interfere with the Rho–mRNA
interactions, blocking termination of transcription as a
consequence. NusA influences both pausing and Rhodependent termination at l tR1, by interaction with the Cterminal domain of the a subunit of E. coli RNA polymerase
(Kainz & Gourse, 1998); however, a direct correlation has
not been established, in this system, between the efficiency
of Rho-dependent termination and the extent of pausing,
two processes that appear to relate to each other in a
complex manner.
Burns et al. (1998) tested the combinatorial effects of NusA
and NusG on transcription elongation and on Rhodependent termination at the tiZ1 and tiZ2 intragenic
terminators, and it seems that the two factors act independently and at different sites: NusG enhances elongation and
Rho-dependent termination, while NusA decreases both,
and these opposing effects are balanced when both factors
act. It would be interesting to know whether NusA and
NusG affect the structural features of the transcription
complex at a pause site.
Models for Rho-dependent termination
Models have been proposed to explain how Rho reaches the
elongation complex stalled at a site downstream from the
initial Rho loading site on the mRNA substrate, and how
the nascent transcript is released. The possibility that Rho
acts at a distance, simply by looping of the RNA, has been
ruled out (Steinmetz et al., 1990), demanding that Rho
translocates from the loading site to the termination site.
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2523
M. S. Ciampi
A physical model for the mechanism of translocation of Rho
59 to 39 along the mRNA was proposed by Geiselmann et al.
(1993). This model, defined as the ‘pure’ tracking model, is a
modification of the simple tracking model (Richardson,
1982), and it predicts that Rho detaches from the initial rut
site of interaction to track 59 to 39 along the RNA chain to
downstream sites, where the nascent RNA is released by the
59 to 39 RNA–DNA helicase activity of Rho (Geiselmann
et al., 1993).
Later studies by Horiguchi et al. (1997) on the quaternary
geometry of Rho factor highlighted structural resemblances
between Rho and other helicases, such as RuvB and T7gp4.
Thus, the finding that RuvB and T7gp4 interact with the
RNA chain within the hole of the hexameric ring led Miwa
et al. (1995) to speculate that the lining of the hole is a site of
interaction with the RNA also for Rho, by analogy with the
RuvB and T7gp4 helicases. This hypothesis was supported
by the finding that rho mutations affecting the interaction of
Rho with the RNA fall within the portion of the protein
participating in the tertiary structure of the ATP-binding
domain of Rho, and extending into the hole (Miwa et al.,
1995). This finding, together with the possibility that Rho
binding to the mRNA occurs by wrapping of the RNA chain
around the N-terminal RNA-binding domains, located on
one side of the Rho hexameric ring, led Horiguchi et al.
(1997) to propose that Rho hooks the RNA through its
N-terminal domain, and that interaction of the RNA with
the central hole allows traction of the RNA.
A similar model had been proposed by Richardson &
Richardson (1996), who suggested that, after the initial
interaction of the 59 end of the Rho utilization site of a
nascent transcript with the RNA-binding domain surface of
Rho, a dissociation and reassociation of subunits would
drive the 39 tail of the rut region of the transcript into the
hole. This would be made possible by a break in the ring,
observed in electron micrographs of unbound Rho (Gogol
et al., 1991). After capturing of the RNA in the hole, Rho
would move 59 to 39 along the RNA, with the aid of ATP
hydrolysis, which provides the energy for dissociation of the
transcript from the RNA polymerase.
The models proposed by Richardson & Richardson (1996)
and by Horiguchi et al. (1997) assign a role to a part of Rho,
the central hole, not previously implicated in translocation
of the protein along the RNA, and they remind one of
another major mode of translocation suggested by studies of
Faus & Richardson (1990) and of Steinmetz & Platt (1994),
and known as the ‘tethered tracking’ mechanism. In this
model Rho is thought to remain bound to the rut site while
translocating 59 to 39 along the mRNA, until it reaches the
actual termination sites. The translocation would be mediated by interaction of the RNA with a secondary site –
proposed by Richardson (1982) – separate from the primary
rut-binding site.
The points of interaction of Rho with RNA were investigated
by Burgess & Richardson (2001) and by Wei & Richardson
2524
(2001). Burgess & Richardson (2001) made Rho proteins
with single cysteine residues, which where photo-crosslinked to various bound derivatives of l cro RNA. Wei &
Richardson (2001) explored the sites of Rho–RNA binding,
through cleavage protection studies in the presence of
bound ATP, RNA or DNA. These studies verified the
existence of a secondary RNA-binding site on the Q-loop of
Rho’s ATP-binding domain, inside the central hole of the
Rho hexamer. The results of Burgess & Richardson (2001)
and of Wei & Richardson (2001) are consistent with binding
of the rut region of the RNA around the crown of the Rho
hexamer and with interaction of the 39side of rut with the
central hole, for activation of ATP hydrolysis and hence
translocation. More recently Skordalakes & Berger (2003)
determined the crystal structure of Rho bound to an ssDNA
recognition site mimic or to an ssRNA and an ATP analogue. These structures show that Rho forms hexameric rings,
with the primary RNA-binding sites facing the inside of the
ring and leading the RNA 39 side toward the hole of the
hexamer. The structures also show that the Rho ring is stably
open in the complexed state. These structural features clarify
how Rho captures the 39 side of the mRNA in the interior of
the ring, before translocation starts.
Conclusions
Rho-dependent transcription termination may be widespread, but most Rho-dependent terminators characterized
occur in enteric bacteria. Known examples of genes regulated by Rho in Gram-positive bacteria are the rho and trp
genes of B. subtilis. Rho-dependent terminators lie in regulatory regions preceding genes, within coding sequences and
at the end of transcriptional units. Some Rho-dependent
terminators are constitutive, but most identified so far are
regulated. This suggests that Rho-dependent termination,
being a complex process involving transcription and translation elongation, is subject to control in many ways, allowing mechanisms to evolve to regulate termination by Rho,
in response to a variety of conditions.
Rho is an RNA-binding protein with ATPase and helicase
activities. It is an essential protein in E. coli and inessential in
B. subtilis, but the two factors function similarly; therefore,
the wider range of rho mutations one could isolate in B.
subtilis might illuminate Rho’s function in E. coli and likely
in S. typhimurium as well. Two additional factors, NusG
and NusA, participate in Rho-dependent termination, by
coupling Rho to the elongation complex. NusG enhances
the rate of release of the RNA from the transcription
elongation complex, and it may mediate Rho action on RNA
polymerase, leading to termination of transcription. NusA
enhances pausing of RNA polymerase, and it may participate in termination by lowering the rate of transcription
elongation. NusA is essential in B. subtilis for reasons that
seem to only partially apply to E. coli. The protein may be
a key to understanding the differences in transcription
elongation and Rho-dependent termination between these
two bacteria, and ultimately to understanding the Rhodependent termination mechanism. Indications of possible
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Rho-dependent terminators and transcription termination
effects of NusG and NusA on the structure of the
transcription complex would illuminate how these factors
mediate Rho action.
Briani, F., Ghisotti, D. & Dehò, G. (2000). Antisense RNA-dependent
transcription termination sites that modulate lysogenic development
of satellite phage P4. Mol Microbiol 36, 1124–1134.
Bult, C. J., White, O., Olsen, G. J. & 20 other authors (1996). Com-
The RNA transcript plays a key role in Rho function, by
mediating interaction between Rho and the transcription
elongation complex. Rho binding to rut on the mRNA
activates Rho’s RNA-dependent ATPase, which drives
Rho 59 to 39 along the message, and the ATP-dependent
RNA–DNA helicase, which ‘peels’ the transcript from its
template. Rho-dependent terminators show a high content
of C residues, required for activation of the Rho ATPase, and
few Gs, minimizing RNA secondary structure and permitting Rho loading onto the transcript. These general features
may be the only elements required for Rho–RNA binding on
naked RNAs of appropriate length, even though specific
nucleotides have been identified at some Rho-dependent
terminators. Other terminator-specific cis-acting sequences,
termed ‘auxiliary elements’ in this review, may aid in Rho
loading, contributing to the efficiency of termination.
Models have been proposed to explain the mode of Rho–
RNA interaction leading to termination of transcription.
Rho-dependent termination is accomplished by the initial
binding of Rho to rut, through the primary RNA-binding
sites of the N-terminal domains of the Rho hexameric
ring; subsequent sliding of the RNA into the central hole of
the hexamer would then occur through a gap in the ring.
Traction would translocate Rho 59 to 39 toward the RNA
termination and release sites, while maintaining association
with the initial binding site, as predicted by the ‘tethered
tracking’ model, or following release of the initial binding
site. Future structural studies will be needed to distinguish
between these two models.
plete genome sequence of the methanogenic archaeon Methanococcus
jannaschii. Science 273, 1058–1072.
Burgess, B. R. & Richardson, J. P. (2001). RNA passes through the
hole of the protein hexamer in the complex with the Escherichia coli
Rho factor. J Biol Chem 276, 4182–4189.
Burns, C. M., Richardson, L. V. & Richardson, J. P. (1998). Com-
binatorial effects of NusA and NusG on transcription elongation
and Rho-dependent termination in Escherichia coli. J Mol Biol 278,
307–316.
Burns, C. M., Nowatzke, W. L. & Richardson, J. P. (1999). Activation
of Rho-dependent transcription termination by NusG. Dependence
on terminator location and acceleration of RNA release. J Biol Chem
274, 5245–5251.
Burova, E., Hung, S. C., Sagitov, V., Stitt, B. L. & Gottesman, M. E.
(1995). Escherichia coli NusG protein stimulates transcription
elongation rates in vivo and in vitro. J Bacteriol 177, 1388–1392.
Calva, E. & Burgess, R. R. (1980). Characterization of a Rho-
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Carlomagno, M. S. & Nappo, A. (2001). The antiterminator NusB
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19–28.
Carlomagno, M. S. & Nappo, A. (2003). NusA modulates intragenic
termination by different pathways. Gene 308, 115–128.
Chen, C. Y. A. & Richardson, J. P. (1987). Sequence elements essential
for r-dependent transcription termination at l tR1. J Biol Chem 262,
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Ciampi, M. S. & Roth, J. R. (1988). Polarity effects in the hisG gene of
Salmonella require a site within the coding sequence. Genetics 118,
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Acknowledgements
Ciampi, M. S., Alifano, P., Nappo, A. G., Bruni, C. B. & Carlomagno,
M. S. (1989). Features of the Rho-dependent transcription termina-
I am grateful to Glenn Herrick for many helpful discussions and for
useful criticisms on earlier versions of this manuscript. I thank an
anonymous referee for constructive comments. I apologize for not
citing all relevant primary papers; this was not possible, due to space
limitations. This article is dedicated to John R. Roth, who inspired it.
tion polar element within the hisG cistron of Salmonella typhimurium. J Bacteriol 171, 4472–4478.
Colonna, B. & Hofnung, M. (1981). rho mutations restore lamB
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